Inflammation is a beneficial response in limiting cellular and organ damages to protect a body from an infection or to remove the damaged tissues, and that a breakdown in the regulation of the inflammatory responses may result in a wide range of inflammatory disorders. Innate immune cells immediately respond to initiate the inflammatory responses to microbial infection and cellular injury, and the responses can be prolonged for duration of time. Sustained cytokine production by macrophages contributes to the prolonged inflammation in vivo. Our preliminary study shows that induction of cell surface 4-1BB ligand (4-1BBL) is essentially required for the sustained TNF production in macrophages. This signaling mechanism occurs sequentially; 4-1BBL is induced in TLR pathway-dependent manner at the initial phase and translocates to the cell surface to form a complex with TLRs at the later phase to activate downstream signaling. Inhibition of the later phase secondary signaling pathway resulted in the reduction of inflammatory responses, and this indicates that 4-1BBL signaling is the potential target of anti-inflammation treatment. However, there are still missing steps in the initial and secondary signaling pathways which control TNF production and its relevance in inflammatory disorder is unclear. We therefore hypothesized that 4-1BBL, expression of which is mediated by TLR-specific signaling pathways, induces a novel second signaling mechanism to sustain the inflammatory response. We propose following specific aims to test our hypothesis. In Aim 1, we will investigate the signaling mechanism of 4-1BBL-mediated TNF production focusing such as the involvement and interaction of signaling molecules in 4-1BBL-mediated secondary signaling pathway. In Aim 2, we will investigate the expression mechanism of 4-1BBL in TLR-mediated signaling pathway to define the specificity of 4-1BBL in inflammatory responses in TLR signaling. Aim 3 is focused on the suppression of inflammatory responses by blocking 4- 1BBL signaling by targeting the 4-1BBL-mediated signaling to reduce the sustained inflammatory response. The studies in this proposal will answer the fundamental question of how inflammation is regulated. The knowledge obtained from these studies will be extended to biological studies and will help to develop new strategies of novel anti-inflammatory therapeutics.